Fermentation with cyclic pulse-pause feeding

ABSTRACT

A process for the production of a valuable compound, comprising the steps of a) fermentation of a filamentous bacterial or fungal strain (e.g. a  Streptomyces  strain or an  Aspergillus  strain) in a fermentation medium wherein a carbohydrate during fermentation is added in a cyclic pulse dosing/pause way, wherein the pulse dosing time is shorter than the pause time and b) recovery of the valuable compound from the fermentation broth.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national application ofPCT/DK2002/000377 filed Jun. 4, 2002, which claims the benefit under 35U.S.C. 119 of U.S. provisional application No. 60/326,611 filed Oct. 1,2001, the contents of which are fully incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a method of reducing broth viscosityduring fermentation.

BACKGROUND ART

Filamentous microorganisms are one of the workhorses for industrialmicrobiology, as they are used for the commercial production of manydifferent therapeutics (e.g. penicillin and cephalosporin), commoditychemicals (e.g. citric acid) and commercial enzymes (e.g. proteases andamylases).

It has been known for decades that fermentations of filamentousmicroorganisms present unique engineering challenges. Specifically, themycelial morphology of filamentous microorganisms often leads to highviscosities which reduce the ability to agitate, pump, and supply oxygento these broths.

Despite extensive study, there has been relatively little success inaltering the morphology to reduce broth viscosity in industrial-scalesystems. In fact, the most common approaches to reduce the brothviscosity have been to add water to dilute the broth or to increaseagitation to fragment the mycelia. Neither of these methods has provento be consistently effective.

SUMMARY OF THE INVENTION

The inventors have found that broth viscosity may be altered in abeneficial way by adjusting the carbon feed profile during fermentationso we claim:

A process for the production of a valuable compound, comprising thesteps of:

a) fermentation of a filamentous bacterial or fungal strain in afermentation medium wherein a carbohydrate during fermentation is addedin a cyclic pulse dosing/pause way, wherein the pulse dosing time isshorter than the pause time; and

b) recovery of the valuable compound from the fermentation broth.

BRIEF DESCRIPTION OF DRAWING

The present invention is further illustrated by reference to theaccompanying drawing, in which

FIG. 1 shows the enzyme activity, the dry cell weight and the brothviscosity (in a normalized form) from 3 fed batch fermentations of aStreptomyces strain (see Example 2). The only difference between thefermentations is that one of them has a prolonged pause time.

DETAILED DISCLOSURE OF THE INVENTION

The inventors have shown that cyclic feeding of cells during fed-batchfermentation may be used as a means to reduce broth viscosity. Duringthe fermentations, glucose was fed either continuously, or in repeated300 sec cycles, with the feed pump on for either 30 or 150 sec duringeach cycle. In all fermentations, cultures were fed the same totalamount of glucose (see Example 1).

Data indicate that pulsed feeding has no significant effect on timeprofiles for total dry cell weight, oxygen mass transfer rate, or totalbase added during the course of each fermentation (variables indicativeof cellular metabolic activity). In addition, pulsed feeding appears tohave no measurable effect on total extracellular protein concentrationor the apparent distribution of extracellular proteins.

In contrast, pulsed feeding has a significant effect on morphology.Cells fed in pulses were smaller than cells fed continuously. As aresult, viscosity is lower in pulse-fed fermentations than infermentations fed continuously.

Valuable Compounds

The valuable compound according to the invention may be an antibioticsuch as penicillin or cephalosporin or erythromycin, or a commoditychemical such as citric acid. The valuable compound may also be atherapeutic protein such as insulin or an enzyme (e.g. a hydrolase, atransferase, a lyase, an isomerase, or a ligase, in particular acarbohydrolase, a cellulase, an oxidoreductase, a protease, an amylase,a lipase, or a carbohydrase).

Microbial Strains

The microbial strain according to the invention may be obtained from anyfilamentous bacterial or fungal strain of any genus.

For example, in a preferred embodiment the valuable compound may beobtained from a Streptomyces strain, e.g., a Streptomyces lividansstrain or Streptomyces murinus strain or from an Actinomyces strain.

In another preferred embodiment the valuable compound may be obtainedfrom a filamentous fungal strain such as an Acremonium, Aspergillus,Aureobasidium, Cryptococcus, Filibasidium, Fusarnum, Humicola,Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora,Paecilomyces, Penicillium, Piromyces, Schizophyllum, Talaromyces,Thernoascus, Thielavia, Tolypocladium, or Trichoderma strain, inparticular the valuable compound may be obtained from an Aspergillusaculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, Fusarium venenatum, Humicola insolens,Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila,Neurospora crassa, Penicillium purpurogenum, Trichoderma harzianum,Trichoderma koningii, Trichoderma iongibrachiatum, Trichoderma reesei,or Trichoderma viride strain.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

For purposes of the present invention, the term “obtained from” as usedherein in connection with a given source shall mean that the valuablecompound is produced by the source or by a cell in which a gene from thesource has been inserted.

Fermentations

The microbial strain may be fermented by any method known in the art.The fermentation medium may be a complex medium comprising complexnitrogen and/or carbon sources, such as soybean meal, cotton seed meal,corn steep liquor, yeast extract, casein hydrolysate, molasses, and thelike. The fermentation medium may be a chemically defined media, e.g. asdefined in WO 98/37179.

The fermentation may be performed as a fed-batch, a repeated fed-batchor a continuous fermentation process.

In a fed-batch process, either none or some of the compounds comprisingone or more of the structural and/or catalytic elements are added to themedium before the start of the fermentation and either all or theremaining part, respectively, of the compounds comprising one or more ofthe structural and/or catalytic elements is fed during the fermentationprocess. The compounds which are selected for feeding can be fedtogether or separate from each other to the fermentation process.

In a repeated fed-batch or a continuous fermentation process, thecomplete start medium is additionally fed during fermentation. The startmedium can be fed together with or separate from the structural elementfeed(s). In a repeated fed-batch process, part of the fermentation brothcomprising the biomass is removed at regular time intervals, whereas ina continuous process, the removal of part of the fermentation brothoccurs continuously. The fermentation process is thereby replenishedwith a portion of fresh medium corresponding to the amount of withdrawnfermentation broth.

In a preferred embodiment of the invention, a fed-batch or a repeatedfed-batch process is applied.

Carbohydrates

Any carbohydrate as defined in Morrison and Boyd: Organic Chemistry, p.1056, 4^(th) edition: “Carbohydrates are polyhydroxy aldehydes,polyhydroxy ketones, or compounds that can be hydrolysed to them. Acarbohydrate that cannot be hydrolysed to simpler compounds is called amonosaccharide. A carbohydrate that can be hydrolysed to twomonosaccharide molecules is called a disaccharide. A carbohydrate thatcan be hydrolysed to many monosaccharides is called a polysaccharide”,may be used according to the invention.

A carbohydrate selected from the group consisting of glucose, sucrose,glucose syrup, fructose, maltose, lactose, trehalose, oligosaccharides,limit dextrins, dextrins, hydrolysed corn dextrin, starch,cyclodextrins, maltulose, mannose, and galactose, is preferred; inparticular a carbohydrate from the group consisting of glucose, sucrose,maltose and hydrolysed dextrin, is preferred.

According to the invention the carbohydrate will normally be added in anamount of from 0.01 g carbohydrate/kg broth/hr to 10 g carbohydrate/kgbroth/hr; in particular in an amount of from 0.1 g carbohydrate/kgbroth/hr to 5 g carbohydrate/kg broth/hr; in a most preferred embodimentof from 0.5 g carbohydrate/kg broth/hr to 2 g carbohydrate/kg broth/hr.

Cyclic Pulse Dosing/Pause

According to the invention the carbohydrate during fermentation is addedin a cyclic pulse dosing/pause way. By “pulsing” the carbohydrate and“pausing” the carbohydrate a “controlled” starvation occurs, and theviscosity is reduced. The overall average feed rate is maintained byincreasing the amount of carbohydrate dosed during the pulse time,according to the ratio of the pulse/pause times.

In a preferred embodiment the carbohydrate pulse dosing is lasting offrom 10 sec. to 1000 sec.; in particular of from 1 sec. to 500 sec.;preferably of from 5 sec. to 100 sec.

In a preferred embodiment the pause is lasting of from 60 sec. to 1800sec. (1 min. to 30 min.); preferably of from 300 sec. to 1800 sec. (5min. to 30 min.).

According to the present invention the pulse dosing time is shorter thanthe pause time, e.g. the pulse dosing time may be lasting 30 sec. andthe pause time may be lasting e.g. 300 sec.

Recovery of the Valuable Compound

A further aspect of the invention concerns the downstream processing ofthe fermentation broth. After the fermentation process is ended, thevaluable compound may be recovered from the fermentation broth, usingstandard technology developed for the valuable compound of interest. Therelevant downstream processing technology to be applied depends on thenature and the cellular localization of the valuable product. First thevaluable compound is separated from the fermentation fluid using e.g.centrifugation or filtration. The valuable compound is recovered fromthe biomass, in case that the valuable product is accumulated inside orassociated with the microbial cells. Otherwise, when the valuableproduct is excreted from the microbial cell, it is recovered from thefermentation fluid as known in the art.

The invention is further illustrated in the following examples which arenot intended to be in any way limiting to the scope of the invention asclaimed.

Example 1 Materials and Methods

Strain and Growth Conditions

In all experiments Aspergillus oryzae was used (derived from strain IFO4177, institute for fermentation, Osaka, Japan).

For storage, freeze-dried spores were suspended with 0.1% Tween 80solution and glycerol was added to a final concentration of 30% (w/v).The spore suspension was maintained at −70° C. For inoculation, frozenspores were germinated on fresh agarose slants (yeast extract 4.0 g/l,Dextrose monohydrate 5.0 g/l, Potassium phosphate monobasic 1.0 g/l,Magnesium sulfate 0.5 g/l, agar 10 g/l), allowed to sporulate, and usedto inoculate seed fermentors (20 L).

In all seed cultures, 8 L of a complex growth medium was used with thefollowing composition: Glucose 20.0 g/l, (NH4)2SO4 2.5 g/l, Yeastextract 10.0 g/l, KH2PO4 1.5 g/l, NaCl 1.0 g/l, MgSO4.7H2O 1.0 g/l,CaCl2.2H2O 0.10 g/l. After sterilization, 1.0 ml of a filter steriletrace mineral solution (ZnSO4.7H2O 5.7 g/l, CuSO4.5H2O 1.0 g/l,NiCl2.6H2O 0.2 g/l, FeSO4.7H2O 5.5 g/l, MnSO4.H2O 3.4 g/l) was added.Medium pH was then adjusted to 3.3 using KOH or H3PO4. During seedfermentations, temperature was maintained at 30° C., air flow rate wascontrolled at 1.0 VVM, impeller speed was controlled at 750 rpm, and pHwas maintained at 3.3 by addition of NH3. Seed culture was grown untiloxygen uptake rate for the cells reached an arbitrary value of 0.3mmol/(liter×min), at which time 5% (v/v) seed culture was used toinoculate experimental fermentations.

Fermentation Conditions

Fermentors with a nominal volume of 20 liters and a working volume of 13liters were used. Growth medium contained: Glucose 5.0 g/l, (NH4)2SO42.5 g/l, KH2PO4 3.75 g/l, NaCl 2.5 g/l, MgSO4.7H2O 2.5 g/l, CaCl2.2H2O0.25 g/l. After sterilization, 17.5 mL filter sterile trace mineralsolution (described above) was added. For all runs, pH was maintained at6.0 using NH3, temperature was maintained at 30° C., air flow rate wascontrolled at 1.0 VVM, and impeller speed was controlled at 750 rpm. Incontrol fermentations, a glucose solution (65% w/v) was fed continuouslyat a rate of 10 g glucose per hr, after a 10% rise in the dissolvedoxygen level during the initial batch mode. Other fermentations were fedas described in the text. Samples were taken at regular intervals andanalyzed for biomass using duplicate measurements of dry cell mass.Morphology and rheological properties were determined as describedbelow.

Morphology

Images of fungal elements, which included both freely dispersed myceliumand clumps, were analyzed in order to quantify morphology. Samples forimage analysis were prepared by mixing 1 ml of broth with equal volumeof fixative solution (Paul, G C and Thomas, C R (1998) Characterizationof Mycelial Morphology Using Image Analysis. Adv. Biochem. Eng. 60:1-59) and stored at 4° C. for later analysis. For image analysis, fixedsamples were diluted with 20% sucrose solution to a final concentrationof 0.2 g/l to prevent artifacts from cell overlap. Images were capturedusing a CCD video camera (Sony) mounted on an inverted stage phasecontrast microscope (IMT-2, Olympus) and digitized by a frame grabbercard (G-3, Scion) installed on a Macintosh computer (Quadra 950). Imageanalysis was done using NIH Image V1.6 downloaded from Internet athttp://rsb.info.nih.gov/nih-image/. Clumps and freely dispersed myceliumwere measured together using average projected area. Since mycelia haveapproximately constant hyphal width, projected area is a close measureof volume and thus can be used to quantify biomass. For each sample,images of at least 100 fungal elements were analyzed to determineaverage projected area for that sample.

Rheological Analysis

All rheological tests were performed using a rotational viscometer(DVII+, Brookfield) with a “vane and cup” geometry. The vane and cupsystem, its calibration, and the rheological testing procedure used havebeen described previously (Marten, M R, Wenger, K S and Khan, S A(1997). Rheology, Mixing Time, and Regime Analysis for aProduction-Scale Aspergillus oryzae Fermentation. Bioreactor andBioprocess Fluid Dynamics. A. W. Nienow. Ednburgh, BHR Group, Cranfield,UK: 295-313). The Herschel-Bulkley equation (τ=τ_(y)+Kγ^(n)) was used todescribe rheological character of all batches, and apparent viscosity(κ) is calculated as (τ_(avg)/γ_(avg)), with ave rage shear stress andshear rate determined as described in Marten, M R, Wenger, K S and Khan,S A (1997). Rheology, Mixing Time, and Regime Analysis for aProduction-Scale Aspergillus oryzae Fermentation. Bioreactor andBioprocess Fluid Dynamics. A. W. Nienow. Ednburgh, BHR Group, Cranfield,UK: 295-313.

Statistical Data Analysis

Analysis of variance (ANOVA) was preformed for statistical comparisons.Significance level (α) was chosen to be 0.05. Thus, a P-value orsignificance probability (P) less than 0.05 is considered an indication(95% confidence) of a significant difference between groups. Toleranceson average values are reported as standard error on the mean.

Gel Electrophoresis

To determine the distribution of the proteins secreted in theextra-cellular medium, SDS-polyacrylamide gels (12% homogeneous,Bio-Rad) were used.

Samples were prepared in Laemmli buffer with β-mercaptoethanol, and wereheated before loading equal volumes. To determine the molecular weightof proteins, low-range molecular weight standard proteins (Bio-Rad) wereused. To quantify protein bands, gels were scanned (GS-800, Bio-Rad) andbands were analyzed (Quantity One software, Bio-Rad) by calculating thetrace quantity (i.e. quantity of a band as measured by the area underits peak profile).

Results

A series of nine fed-batch fermentations at three different “pulsefraction” (PF) values were conducted:Pulse Fraction (PF)=(Feed−Pump “On” Time)/(Total Cycle Time).

In all the fermentations total cycle time was 300 sec. As a control,three fed-batch fermentations were carried out with PF=1.0, and aconstant glucose feed rate of 10 g/hr. A second set of threefermentations was carried out with PF=0.5, and glucose addition rate of20 g/hr, and a third set of three fermentations was conducted withPF=0.1, and glucose feed rate of 100 g/hr. With this arrangement, thesame total amount of glucose was added in all fermentations, regardlessof the PF value, during each five minute cycle. Initial glucoseconcentration during all fermentations was 5 g/l, and feeding startedafter a 10% rise in dissolved oxygen. On-line measurements were made foroxygen uptake rate (OUR), carbon dioxide evolution rate (CER), totalbase added, and samples were taken for off-line analysis of fungalmorphology, broth rheology and biomass concentration.

Fungal biomass, measured as dry cell weight, increases approximatelylinearly over the course of all fermentations to a final value ofapproximately 17 g/l. A regression analysis showed no significantdifference (95% confidence) between biomass profiles for fermentationsat the three different PF values.

Total base added for pH control rised approximately linearly for allbatches with no discernable difference for the three PF values. Thus,pulsed feeding during fed-batch operation has no apparent effect onthese variables, indicative of cellular metabolic activity.

Total extra-cellular protein concentration as a function of time duringthe same nine fermentations showed that the initial proteinconcentration of approximately 1 g/l, due to carry-over of protein fromthe inoculum, failed during the batch portion of the fermentation. Afterinitial glucose was consumed, feeding begins and low residual glucoselevels allow expression of secreted proteins. This leads to theapparently linear rise in total protein concentration that continuesuntil the end of each batch. A regression analysis showed no significantdifference (95% confidence) between protein concentration profiles forfermentations at the three different PF values.

Extra-cellular protein concentration (g extra-cellular protein per g drycell mass) during the fed-batch portion of the fermentations showedaverage values of 0.11±0.003, 0.11±0.005 and 0.10±0.005 for PF values of1.0, 0.5 and 0.1 respectively, with no significant difference betweenthese values. Thus, it appears that pulsed feeding has no observableeffect on total extra-cellular protein concentration profiles.

In addition to total extra-cellular protein, we used SDS-PAGE to followthe apparent distribution of these proteins as a function of time. Therelative concentration of six of these proteins was determined, andregression analysis showed no significant difference in the apparentdistribution of proteins between fermentations operated at the differentPF values. Thus, pulsed feeding does not appear to have changed theexpression pattern or apparent distribution of extracellular proteins inthis system.

While pulse feeding had no observable effect on metabolic variables orextra-cellular protein expression, it had a measurable effect on fungalmorphology, in particular, the average size of fungal elements. Tomeasure the size of fungal elements we use average projected area (A),which takes into account both freely dispersed mycelia and clumps (nopellets were found in any fermentations described here). The averageprojected area can be divided into three distinct time periods. Duringthe first period or batch portion of each fermentation (t<18 hr), Arises as a function of time. During the second period (18<t<50 hr),initial glucose is exhausted, feeding has begun, and A begins todecrease. This continues until the third period (50<t<110 hr), where Aremains at an approximately constant value. Regression analysis shows nosignificant difference between A profiles during these first twoperiods. In contrast, between 50 hours and the end of the each batch,there is a significant difference in time averaged A betweenfermentations with PF=1.0 and 0.5 (P=2.2×10−4), and betweenfermentations with PF=1.0 and 0.1 (P=7.4×10−5).

Thus, pulsed feeding had a measurable and significant effect on fungalmorphology. Fungal elements in pulsed fermentations were smaller thanthose fed a continuous stream of glucose. Morphological behavior duringthese fermentations appears to have had a measurable effect on brothviscosity. We found that just as with morphology, behavior of brothviscosity can be divided into three distinct time periods. During thefirst period (t<18 hr) viscosity rose as a function of time, during thesecond period (180<t<50 hr) viscosity remained relatively constant, andduring the third period (t>50 hr) viscosity rose again.

During the first time period or batch phase, both biomass and A wereincreasing, and as a result viscosity also increased. During the secondperiod, biomass continued to increase, but A was decreasing. Apparently,these two phenomena off-set each other, and as a result viscosityremained relatively constant. During the third time period, biomass rosewhile A remained constant, leading to a second rise in viscosity.Statistical analysis of time averaged viscosity during the first twoperiods (t<50 hr) showed no significant difference for fermentationsoperated at the three different PF values. However, during the thirdperiod (t>50 hr) viscosity in pulse-fed fermentations was significantlylower than viscosity in control fermentations. We found that the timeaveraged values of viscosity during this period were 0.47±0.073,0.23±0.022, and 0.17±0.019 for fermentations operated at PF values of1.0, 0.5, and 0.1 respectively, with a statistically significantdifference between fermentations operated at PF=1.0 and 0.5(P=1.8×10⁻⁵), and between fermentations operated at PF=1.0 and 0.1(P=2.4×10⁻⁶).

It appears that a simple feeding strategy, in particular wherein thepulse dosing time is shorter than the pause time, can be used to producesmaller fungal mycelia, leading to a significant reduction infermentation broth viscosity.

Example 2 Streptomyces Fermentation

The second example of a beneficial reduction in broth viscosity comesfrom the filamentous bacterium Streptomyces murinus, producing a nativeintracellular protein. Prolonging the pause time increases productivityslightly and significantly reduces the broth viscosity.

Method

Three identical pilot scale fermenters were seeded from the same seedtank and ran simultaneously with a start weight of 250 kg for equallengths of time. The seed medium was formulated from common fermentationingredients including glucose, corn-steep liquor, potassium phosphateand ammonium sulphate. The main tank medium was similarly formulated andalso included sodium phosphate, magnesium sulphate and some tracemetals. After an initial period of growth in the main tank, a glucosefeed was started and the rate held constant for the rest of thefermentation. I.e. the fermentation was typical of a fermentationprocess for this sort of organism.

Measurements of Enzyme activity per unit weight of culture broth, anddry cell weight per unit volume of culture broth were measured byvalidated methods conforming with ISO 9001. Viscosity was measured in aCarrimed controlled stress rheometer with a 6 cm cone and plategeometry. The instrument was instructed to apply a shear rate of 10 s⁻¹& 100 s⁻¹ and the steady state stress used to calculate the apparentviscosity.

Results

FIG. 1 shows the results (in a normalized form) from the three fed batchfermentations. The only difference between the fermentations is that thelast of the three ran with a prolonged pause time: The pulse dosing timewas as fast as possible (<10 sec.), and the pause time was 7 min.

The activity per unit mass data shows a small but positive effect on theenzyme activity from pulse-paused dosing. The cell dry weight per unitvolume data shows no effect from pulse-paused dosing. The viscosity datashows a dramatic and highly beneficial reduction in broth viscosity uponfor pulse paused dosing.

CONCLUSION

Pulse-Paused dosing results in a small increase in productivity and adramatic reduction of viscosity. This has a major impact on mixingperformance at the large scale and will lead to the opportunity ofrunning large scale fermentations to a higher biomass concentration andtherefore a higher productivity.

1. A process for producing a compound, comprising the steps of: (a)fermenting a filamentous bacterial or fungal strain in a fermentationmedium wherein a carbohydrate is added during fermentation to thefermentation medium by a cyclic pulse dosing/pause method, wherein thecyclic pulse dosing/pause method comprises alternating between (i)dosing the fermentation medium with the carbohydrate for a pulse feedingperiod of from 1 to 1000 seconds and (ii) a pausing period of from 60 to1800 seconds without dosing the carbohydrate, wherein the carbohydrateis feed for the filamentous bacterial or fungal strain; and (b)recovering the compound from the fermentation medium; wherein thecompound is an antibiotic or a protein.
 2. The process of claim 1,wherein the compound is an antibiotic.
 3. The process of claim 1,wherein the compound is a protein.
 4. The process of claim 3, whereinthe protein is an enzyme.
 5. The process of claim 4, wherein the enzymeis a hydrolase, oxidoreductase, isomerase, ligase, lyase, ortransferase.
 6. The process of claim 5, wherein the enzyme is anamylase, carbohydrase, cellulase, lipase, or protease.
 7. The process ofclaim 1, wherein the fermentation is with a filamentous bacterialstrain.
 8. The process of claim 7, wherein the filamentous bacterialstrain is an Actinomyces or Streptomyces strain.
 9. The process of claim1, wherein the fermentation is with a filamentous fungal strain.
 10. Theprocess of claim 9, wherein the filamentous fungal strain is anAspergillus strain.
 11. The process of claim 10, wherein the filamentousfungal strain is an Aspergillus oryzae strain.
 12. The process of claim1, wherein the carbohydrate is selected from the group consisting ofglucose, sucrose, glucose syrup, fructose, maltose, lactose, trehalose,oligosaccharides, limit dextrins, dextrins, hydrolyzed dextrin, starch,cyclodextrins, maltulose, mannose, and galactose.
 13. The process ofclaim 12, wherein the carbohydrate is selected from the group consistingof glucose, hydrolyzed dextrin, maltose, and sucrose.
 14. The process ofclaim 1, wherein the carbohydrate is added in an amount of from 0.01 gcarbohydrate/kg broth/hr to 10 g carbohydrate/kg broth/hr.
 15. Theprocess of claim 14, wherein the carbohydrate is added in an amount offrom 0.1 g carbohydrate/kg broth/hr to 5 g carbohydrate/kg broth/hr. 16.The process of claim 15, wherein the carbohydrate is added in an amountof from 0.5 g carbohydrate/kg broth/hr to 2 g carbohydrate/kg broth/hr.17. The process of claim 1, wherein the pulse feeding period is from 1to 500 seconds and the pausing period is from 300 to 1800 seconds. 18.The process of claim 17, wherein the pulse feeding period is from 5 to100 seconds.
 19. The process of claim 17, wherein the fermentation is afed-batch or a repeated fed-batch process.
 20. The process of claim 1,wherein the pulse feeding period is shorter than the pausing period. 21.A process for producing a compound, comprising the steps of: (a)fermenting a filamentous bacterial or fungal strain in a fermentationmedium wherein a carbohydrate is added during fermentation to thefermentation medium by a cyclic pulse dosing/pause method, wherein thecyclic pulse dosing/pause method comprises alternating between (i)dosing the fermentation medium with the carbohydrate for a pulse feedingperiod of from 1 to 500 seconds and (ii) a pausing period of from 60 to1800 seconds without dosing the carbohydrate, wherein the carbohydrateis feed for the filamentous bacterial or fungal strain; and (b)recovering the compound from the fermentation medium; wherein thecompound is an antibiotic or a protein.